Visualizing Ghrelin Receptor through Genetically Encoded Labeling for Monitoring the Single-Molecule Conformational Dynamics

The ghrelin receptor (GhR) is a class A G protein-coupled receptor (GPCR) involved in the entero-endocrine signaling systems that regulates food intake and energy homeostasis. The GhR is noted for its unusually high basal constitutively activity. GhR is a potential drug target for “diabesity” syndromes, and the interaction between GhR and its endogenous peptide ligand, ghrelin, has been intensively studied. However, there is only a limited understanding of GhR pharmacology and its molecular mechanism of signal transduction. Using well-established amber codon suppression technology and state-of-the-art single-molecule techniques, we are developing tools to monitor directly differential conformational dynamics of GhR in the presence and absence of its binding partners, including ligands, G proteins, or other GPCRs. For example, we are preparing single-site and double-site fluorescently labeled GhR and a series of labeled ghrelin analogues. These engineered receptors can be studied in cell-based systems or reconstituted in NABBs (Nanoscale Apolipoprotein Bound Bilayers) after purification. These types of approaches will enable us to better understand the complexity of GhR signaling in the neuro-endocrine system, providing insights to design specific drugs for targeting fine-tuned signal pathways involved in metabolic disorders like obesity and diabetes.